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WO2024097608A2 - Peptides et probiotiques ciblés pour la réduction ou l'augmentation de lymphocytes t mémoires à réaction croisée d'auto-antigènes bactériens pour traiter le cancer ou une maladie auto-immune - Google Patents

Peptides et probiotiques ciblés pour la réduction ou l'augmentation de lymphocytes t mémoires à réaction croisée d'auto-antigènes bactériens pour traiter le cancer ou une maladie auto-immune Download PDF

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Publication number
WO2024097608A2
WO2024097608A2 PCT/US2023/078034 US2023078034W WO2024097608A2 WO 2024097608 A2 WO2024097608 A2 WO 2024097608A2 US 2023078034 W US2023078034 W US 2023078034W WO 2024097608 A2 WO2024097608 A2 WO 2024097608A2
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bacteria
composition
cells
tumor
subject
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PCT/US2023/078034
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WO2024097608A3 (fr
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Mary L. Disis
Denise CECIL
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University Of Washington
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/23—Lactobacillus acidophilus

Definitions

  • Th1 T-helper 1 interferon-gamma
  • Th2 IL-10 T-helper-2
  • compositions and methods for enhancing immunity to a tumor antigen in a subject as well as for slowing tumor growth in a subject in need thereof.
  • these methods comprise administering to the subject an effective amount of a composition comprising a plurality of bacteria that exhibit less than 50% amino acid sequence homology to non-mutated tumor antigens (bacteria low ).
  • the plurality of bacteria low are selected from: Lactobacillus acidophilus, Ruminococcus albus, Ruminococcus gnavus, Dorea formicigenerans, Eubacterium siraeum, Bacteroides stercoris, Parabacteroides distasonis, Bifidobacterium breve, Lactobacillus delbrueckii, Odoribacter splanchnicus, Akkermansia muciniphila, Desulfovibrio piger, Mitsuokella multacida, Bifidobacterium catenulatum, Staphylococcus saprophyticus, Bacteroides vulgatus, Bifidobacterium pseudocatenulatum, Bifidobacterium angulatum, Bifidobacterium bifidum, Campylobacter hominis, Clostridium leptum, Clostridium methylpentosum, Coprococcus catus, Eubacterium ventrios
  • the composition is free of bacteria exhibiting greater than 50% homology to tumor antigens identified in a blood sample obtained from the subject.
  • the method further comprises obtaining such a blood sample from the subject, and identifying bacterial-tumor antigen (BAC-TA) cross-reactive memory T-cells detected in the sample.
  • the composition is administered weekly for six weeks. In some embodiments, the composition is administered twice a week. In some embodiments, the composition is administered twice a month.
  • the composition is administered monthly. In some embodiments, the administration continues for at least two months. In some embodiments, the administration continues for at least 6, 8, 12, or 24 months. [0008] In some embodiments, the effective amount is sufficient to effect a two-fold increase in gut microbiome bacteria low , and/or a significant decrease in bacterial-tumor antigen (BAC- TA) cross-reactive memory T-cells, in a biological sample obtained from the subject. In some embodiments, the method enhances efficacy of a cancer treatment. In some embodiments, the cancer treatment comprises immunotherapy and/or chemotherapy. In some embodiments, the immunotherapy comprises administration of immune checkpoint inhibitor therapy, vaccine therapy, cytokine therapy, and/or oncolytic virus therapy.
  • the chemotherapy comprises administration of paclitaxel.
  • Other chemotherapeutic agents, as well as other immunotherapeutic agents, known to those skilled in the art are likewise contemplated.
  • Some representative examples of chemotherapeutic agents include, but are not limited to, cisplatin, carboplatin, pemtrexed, Adriamycin, Cytoxan.
  • Some representative examples of immunotherapeutic agents include, but are not limited to, monoclonal antibodies, such as, for example, anti-HER2, anti-EGFR, and anti-VEGF.
  • the composition is administered prior to and/or concomitantly with immunotherapy or chemotherapy. [0009]
  • the method enhances a type 1 immune response.
  • the method increases the relative amount of bacteria low that can be detected in a biological sample obtained from the subject.
  • biological samples include blood and stool samples.
  • the composition comprises 1-100 x10 9 CFU of bacteria low .
  • the composition comprises 1-50 x10 9 CFU of bacteria low .
  • the composition comprises 1-10 x10 9 CFU of bacteria low .
  • the composition comprises 1-5 x10 9 CFU of bacteria low .
  • the administration of the composition is oral, rectal, or intragastric.
  • the composition is delivered in the form of a suppository.
  • composition comprising a plurality of bacteria that exhibit less than 50% homology to non-mutated tumor antigens (bacteria low ).
  • the plurality of bacteria low are selected from: Lactobacillus acidophilus, Ruminococcus albus, Ruminococcus gnavus, Dorea formicigenerans, Eubacterium siraeum, Bacteroides stercoris, Parabacteroides distasonis, Bifidobacterium breve, Lactobacillus delbrueckii, Odoribacter splanchnicus, Akkermansia muciniphila, Desulfovibrio piger, Mitsuokella multacida, Bifidobacterium catenulatum, Staphylococcus saprophyticus, Bacteroides vulgatus, Bifidobacterium pseudocatenulatum, Bifidobacterium angulatum, Bifidobacterium bifidum
  • the plurality of bacteria low comprise R. albus and L. acidophilus.
  • the composition is substantially free of bacteria that exhibit greater than 50% homology to tumor antigens.
  • the method comprises administering to the subject an effective amount of a composition comprising a plurality of bacteria that exhibit at least 50% (high) homology to self-proteins (bacteria high ).
  • the plurality of bacteria high are selected from: Escherichia coli, Enterococcus faecalis, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas aeruginosa, Shigella dysenteriae, Enterobacter cloacae, Lactococcus lactis, Clostridium perfringens, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecium, Rhodopseudomonas palustris, Shigella boydii, Shigella sonnei, Finegoldia magna, Clostridium asparagiforme, Collinsella aerofaciens, Fusobacterium ulcerans, Aerococcus viridans, Bacteroides ovatus, Clostridium botulinum, Enterococcus gallinarum, Eubacterium limosum, Faecalibacter
  • the plurality of bacteria high exhibit at least 50% homology to self-proteins identified in a blood and/or stool sample obtained from the subject.
  • the composition is administered weekly for six weeks to one year. In some embodiments, the composition is administered weekly for at least one year. In some embodiments, the composition is administered twice a week. In some embodiments, the composition is administered twice a month. In some embodiments, the composition is administered monthly. In some embodiments, the administration continues for at least two months. In some embodiments, the administration continues for at least 6, 8, 12, or 24 months. [0015] In some embodiments, the effective amount is sufficient to effect a two-fold increase in gut microbiome bacteria high in a biological sample obtained from the subject.
  • the method further comprises obtaining a biological sample from the subject, and identifying self-protein reactive memory T-cells.
  • the method elicits a type 2 immune response.
  • the method elicits an increase in the amount of IL-10 and/or IL-6 that can be detected in a biological sample obtained from the subject.
  • the composition 1-100 x10 9 CFU of bacteria high .
  • the composition comprises 1-50 x10 9 CFU of bacteria high .
  • the composition comprises 1-10 x10 9 CFU of bacteria high .
  • the composition comprises 1-5 x10 9 CFU of bacteria high .
  • the administration of the composition is oral, rectal, or intragastric.
  • the composition is delivered in the form of a suppository.
  • a composition comprising a plurality of bacteria that exhibit at least 50% amino acid sequence homology to self-proteins (bacteria high ).
  • the plurality of bacteria high are selected from: Escherichia coli, Enterococcus faecalis, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas aeruginosa, Shigella dysenteriae, Enterobacter cloacae, Lactococcus lactis, Clostridium perfringens, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecium, Rhodopseudomonas palustris, Shigella boydii, Shigella sonnei, Finegoldia magna, Clostridium asparagiforme, Collinsella aerofaciens, Fusobacterium ulcerans, Aerococcus viridans, Bacteroides ovatus, Clostridium botulinum, Enterococcus gallinarum, Eubacterium limosum, Faecalibacter
  • the plurality comprises at least four species of bacteria high . In some embodiments, the plurality comprises 6-10 species of bacteria high .
  • the subject to be treated in the above methods is human. In some embodiments, the subject is suspected of having, or has been diagnosed with cancer, such as breast cancer, or an autoimmune disease. BRIEF DESCRIPTION OF THE DRAWINGS [0022] FIGS.1A-1D show Human T-cell lines generated from IL-10-inducing epitopes are specific for the tumor antigen and cross-reactive to bacteria high . (1A) Epitope induced IL-10- secreting precursor frequency to the indicated epitope presented as box and whisker plots.
  • Each dot is the precursor frequency for an individual donor that demonstrated a significant IL-10 response.
  • Negative controls include an irrelevant peptide (HIVp52) or protein (BSA) or bacteria that shares no sequence homology (R. albus).
  • D Percent change in the indicted memory T-cell subset in the antigen-specific T-cell line compared to the autologous PBMC.
  • FIGS.2A-2D show that BAC-TA cross-reactive T-cells traffic to mammary cancer and promote tumor growth.
  • (2A) Epitope induced IL-10-secreting precursor frequency to the indicated epitope presented as box and whisker plots with Tukey outliers in na ⁇ ve FVB mice (n 10)
  • (2B) Total flux ( ⁇ SEM) of fluorescently labeled T-cells measured in tumors from mice infused with P. aeruginosa-specific or naive T-cells (n 5). Representative fluorescent imaging quantified in panel.
  • FIG.3 shows that tumor infiltrating lymphocytes in spontaneous breast cancer are specific for bacteria high .
  • FIGS.4A-4C show that oral gavage of live bacteria resulted in increased bacterial counts of the gavaged species.
  • 4A Experimental design schematic. Number of bacteria detected in the stool of mice treated with (4B) R. albus and L. Acidophilus or untreated and stool measured for those bacteria (4C) E. coli and P.
  • FIG.5 shows that treatment with L. Acidophilus and R. albus can enhance vaccine immunogenicity.
  • Mean ( ⁇ SEM) IFN-g corrected spots per well (CSPW) for mice immunized with the indicated vaccine and treated with the indicated bacteria. n 8 mice/group; *p ⁇ 0.05.
  • FIGS.6A-6B show that mammary tumors in the C3T are resistant to immunotherapy and chemotherapy.
  • FIGS.7A-7D show that tumor antigen epitopes with elevated IL-10-secreting T-cell precursor frequencies share high sequence homology with epitopes from bacteria in the gut microbiome.
  • FIGS.8A-8B show that there is no significant association between the relative start location or the number of amino acid mutations near an epitope and adjusted IL10 spot count factor of any epitope.
  • a locally estimated scatterplot smoothing (loess) line is drawn to show the smoothed mean and the shaded area corresponds to the 95% confidence interval for the smoothed mean.
  • FIGS.9A-9F show that T-cell lines generated from IL-10-inducing tumor antigen epitopes are cross-reactive to bacteria with high sequence homology, express cytokines and genes associated with intestinal intraepithelial lymphocytes and are of a memory phenotype.
  • ⁇ SD Mean ( ⁇ SD) IL-10 corrected spots per well (CSPW) for T-cell lines specific for (9A) Yb1-p82, (9B) BIRC5-p13 and (9C) PRL3-p104 stimulated with the line-generating epitope, corresponding recombinant protein or bacteria that share >50% sequence homology with the line-generating epitope.
  • Negative controls include an irrelevant peptide (HIVp52) or protein (BSA) or a bacteria that shares no sequence homology (R. Albus).
  • FIGS.10A-10E show that murine T-cell lines generated from the same IL-10- inducing tumor antigen epitopes observed in humans recapitulated the phenotype of the human T-cell lines.
  • Negative controls include an irrelevant peptide (HIVp52) or protein (BSA) or a bacteria that shares no sequence homology (R. Albus).
  • FIGS.11A-11B show that significantly more TCRb were common between intestinal intraepithelial lymphocytes and tumor infiltrating lymphocytes than splenocytes and tumor infiltrating lymphocytes.
  • IEL intestinal intraepithelial lymphocytes
  • TIL tumor infiltrating lymphocytes
  • FIGS.11A-11B show that significantly more TCRb were common between intestinal intraepithelial lymphocytes and tumor infiltrating lymphocytes than splenocytes and tumor infiltrating lymphocytes.
  • IEL intestinal intraepithelial lymphocytes
  • TIL tumor infiltrating lymphocytes
  • spleen right circles
  • Fold change of the number of shared TCRb in IEL, TIL and spleen for each mouse normalized to the number of shared TCRb between TIL and spleen. n 7 mice; ***p ⁇ 0.001.
  • FIGS.12A-12F show that Pseudomonas aeruginosa-specific T-cells traffic to breast cancer and promote tumor growth.
  • (12A Mean ( ⁇ SD) IL-10 corrected spots per well (CSPW) for T-cell lines specific for P. aeruginosa stimulated with the line-generating bacteria, homologous epitope Yb1-p82 and recombinant protein Yb1. Negative controls include an irrelevant peptide (HIVp52) or protein (BSA) or bacteria that is not homologous (R. Albus).
  • (12B Total flux ( ⁇ SEM) of fluorescently labeled T-cells measured in tumors from mice infused with P.
  • aeruginosa-specific or naive T-cells (12C) Representative fluorescent imaging quantified in 12C.
  • Mean ( ⁇ SEM) is shown with horizontal bars for IL-10 CSPW from tumor infiltrating lymphocytes (open circles) or spleen (hatched circles) from C3T mice with spontaneous tumors treated with the indicated bacterial lysate.
  • FIGS.13A-13F show that bacterial-tumor antigen cross reactive-specific TCRb can be identified in tumor biopsies of newly diagnosed breast cancer patients.
  • CSPW IL-10 corrected spots per well
  • 13C Venn diagram for the number of TCRb sequences in the HIF1a-p312-specific T-cell line (left circle) and corresponding tumor biopsy (right circle) from Donor 1.
  • 13D Percent of the total tumor TCRb repertoire for the indicted TCR for Donor 1.
  • lymphocytes are cross-reactive for both bacterial and tumor antigens.
  • BAC-TA bacterial-tumor antigen
  • bacteria with low to no homologies bacteria with low to no homologies (bacteria low ) can be safely enriched in the gut and prevent BAC-TA from expansion. This precision modulation of the gut microbiome reduces tumor growth and increases the efficacy of immune checkpoint inhibitor therapy in a transgenic mouse mammary tumor model of triple negative breast cancer.
  • transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the recited embodiment.
  • the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.” “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions disclosed herein. Aspects defined by each of these transition terms are within the scope of the disclosure herein.
  • a linker refers to an amino acid sequence that connects two proteins, polypeptides, peptides, domains, regions, or motifs and may provide a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity to a target molecule or retains signaling activity.
  • a linker is comprised of about two to about 35 amino acids, for instance, or about four to about 20 amino acids or about eight to about 15 amino acids or about 15 to about 25 amino acids.
  • nucleic acid sequence or “polynucleotide” refers to nucleotides of any length which are deoxynucleotides (i.e. DNAs), or derivatives thereof; ribonucleotides (i.e. RNAs) or derivatives thereof; or peptide nucleic acids (PNAs) or derivatives thereof.
  • the terms include, without limitation, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, oligonucleotides (oligos), or other natural, synthetic, modified, mutated or non-natural forms of DNA or RNA.
  • the term “vector” refers to, without limitation, a recombinant genetic construct or plasmid or expression construct or expression vector that retains the ability to infect and transduce non-dividing and/or slowly-dividing cells and integrate into the target cell’s genome.
  • the vector may be derived from or based on a wild-type virus. Aspects of this disclosure relate to an adeno-associated virus vector, an adenovirus vector, and a lentivirus vector.
  • expression control element refers to any sequence that regulates the expression of a coding sequence, such as a gene.
  • Exemplary expression control elements include but are not limited to promoters, enhancers, microRNAs, post- transcriptional regulatory elements, polyadenylation signal sequences, and introns.
  • Expression control elements may be, without limitation, constitutive, inducible, repressible, or tissue-specific.
  • a “promoter” is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors.
  • expression control by a promoter is tissue-specific.
  • An “enhancer” is a region of DNA that can be bound by activating proteins to increase the likelihood or frequency of transcription.
  • Non-limiting exemplary enhancers and posttranscriptional regulatory elements include the CMV enhancer and WPRE.
  • multicistronic or “polycistronic” or “bicistronic” or tricistronic” refers to mRNA with multiple, i.e., double or triple coding areas or exons, and as such will have the capability to express from mRNA two or more, or three or more, or four or more, etc., proteins from a single construct. Multicistronic vectors simultaneously express two or more separate proteins from the same mRNA.
  • the two strategies most widely used for constructing multicistronic configurations are through the use of 1) an IRES or 2) a 2A self- cleaving site.
  • an “IRES” refers to an internal ribosome entry site or portion thereof of viral, prokaryotic, or eukaryotic origin which are used within polycistronic vector constructs.
  • an IRES is an RNA element that allows for translation initiation in a cap- independent manner.
  • self-cleaving peptides or “sequences encoding self- cleaving peptides” or “2A self-cleaving site” refer to linking sequences which are used within vector constructs to incorporate sites to promote ribosomal skipping and thus to generate two polypeptides from a single promoter, such self-cleaving peptides include without limitation, T2A, and P2A peptides or sequences encoding the self-cleaving peptides.
  • “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules.
  • Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of disclosed herein. [0045] Percent similarity or percent complementary of any of the disclosed sequences may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG).
  • UWGCG University of Wisconsin Genetics Computer Group
  • the GAP program utilizes the alignment method of Needleman and Wunsch (1970). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences.
  • the preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non- identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess (1986), (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
  • Nucleotide sequence refers to a heteropolymer of deoxyribonucleotides, ribonucleotides, or peptide-nucleic acid sequences that may be assembled from smaller fragments, isolated from larger fragments, or chemically synthesized de novo or partially synthesized by combining shorter oligonucleotide linkers, or from a series of oligonucleotides.
  • protein As used herein, the terms “protein”, “peptide”, and “polypeptide” refer to amino acid subunits, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds.
  • the subunit may be linked by other bonds, e.g., ester, ether, etc.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids.
  • recombinant expression system or “recombinant expression vector” refers to a genetic construct for the expression of certain genetic material formed by recombination.
  • effective amount or “therapeutically effective amount” or “prophylactically effective amount”, refer to an amount of an active agent described herein that is effective to provide the desired/intended result and/or biological activity.
  • an effective amount of a composition described herein is an amount that is effective to result in remission or slowing the progression of disease, and/or to improve or to ameliorate symptoms of and/or to treat disease.
  • an equivalent or a biologically equivalent of such is intended within the scope of this disclosure.
  • biological equivalent thereof is intended to be synonymous with “equivalent thereof” when referring to a reference small molecule, polypeptide, protein, polynucleotide, nucleic acid, oligonucleotide, antisense, or miRNA even those reference molecules having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any nucleic acid, polynucleotide, oligonucleotide, antisense, miRNA, polypeptide, or protein mentioned herein also includes equivalents thereof.
  • an equivalent intends at least about 70% homology or identity, or at least 80 % homology or identity and alternatively, or at least about 85 %, or alternatively at least about 90 %, or alternatively at least about 95 %, or alternatively 98 % percent homology or identity and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid.
  • an equivalent thereof is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement.
  • the polypeptide and/or polynucleotide sequences are provided herein for use in gene and protein transfer and expression techniques described herein.
  • Such sequences provided herein can be used to provide the expression product as well as substantially identical sequences that produce a protein that has the same biological properties.
  • These “biologically equivalent” or “biologically active” or “equivalent” polypeptides are encoded by equivalent polynucleotides as described herein. They may possess at least 60%, or alternatively, at least 65%, or alternatively, at least 70%, or alternatively, at least 75%, or alternatively, at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% or alternatively at least 98%, identical primary amino acid sequence to the reference polypeptide when compared using sequence identity methods run under default conditions. Specific polynucleotide or polypeptide sequences are provided as examples of particular embodiments.
  • an equivalent polynucleotide is one that hybridizes under stringent conditions to the reference polynucleotide or its complement or in reference to a polypeptide, a polypeptide encoded by a polynucleotide that hybridizes to the reference encoding polynucleotide under stringent conditions or its complementary strand.
  • an equivalent polypeptide or protein is one that is expressed from an equivalent polynucleotide.
  • “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PC reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
  • Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about 10x SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC.
  • Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC.
  • high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about 0.1x SSC; formamide concentrations of about 55% to about 75%; and wash solutions of about lx SSC, 0.1x SSC, or deionized water.
  • hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes.
  • SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.
  • treating or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • isolated means that a naturally occurring DNA fragment, DNA molecule, coding sequence, or oligonucleotide is removed from its natural environment, or is a synthetic molecule or cloned product.
  • the DNA fragment, DNA molecule, coding sequence, or oligonucleotide is purified, i.e., essentially free from any other DNA fragment, DNA molecule, coding sequence, or oligonucleotide and associated cellular products or other impurities.
  • the term “cell” as used herein refers to either a prokaryotic or eukaryotic cell, optionally obtained from a subject or a commercially available source.
  • Cells treated, transfected, transformed, or otherwise in contact with compositions and/or nucleic acid molecules disclosed herein include without limitation, cells of a human, non-human animal, mammal, or non-human mammal, including without limitation, cells of murine, canine, or non-human primate species.
  • Cells treated, transfected, transformed, or otherwise in contact with compositions and/or nucleic acid molecules disclosed herein are, without limitation, T cells, antigen-presenting cells, and other suitable host cells.
  • the term "subject" includes any human or non-human animal.
  • non-human animal includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects.
  • a or “an” means at least one, unless clearly indicated otherwise.
  • to “prevent” or “protect against” a condition or disease means to hinder, reduce or delay the onset or progression of the condition or disease.
  • encode refers to a polynucleotide which is said to “encode” a polypeptide, an mRNA, or an effector RNA if, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and/or translated to produce the effector RNA, the mRNA, or an mRNA that can for the polypeptide and/or a fragment thereof.
  • the antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
  • expression refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. The expression level of a gene may be determined by measuring the amount of mRNA or protein in a cell or tissue sample; further, the expression level of multiple genes can be determined to establish an expression profile for a particular sample.
  • the term “functional” may be used to modify any molecule, biological, or cellular material to intend that it accomplishes a particular, specified effect.
  • the term “about,” as used herein when referring to a measurable value such as an amount, level or concentration, for example and without limitation, is meant to encompass variations of 20%, 10%, 5%, 1 %, 0.5%, or even 0.1 % of the specified amount, or fold differences in levels of a quantifiable comparison with a standard or control or reference material, such as 1-fold, 2-fold, 3-fold, 4-fold...10-fold, 100-fold, etc. of the specified level of comparison.
  • these methods comprise administering to the subject an effective amount of a composition comprising a plurality of bacteria that exhibit less than 50% amino acid sequence homology to non-mutated tumor antigens (bacteria low ).
  • the plurality of bacteria low are selected from: Lactobacillus acidophilus, Ruminococcus albus, Ruminococcus gnavus, Dorea formicigenerans, Eubacterium siraeum, Bacteroides stercoris, Parabacteroides distasonis, Bifidobacterium breve, Lactobacillus delbrueckii, Odoribacter splanchnicus, Akkermansia muciniphila, Desulfovibrio piger, Mitsuokella multacida, Bifidobacterium catenulatum, Staphylococcus saprophyticus, Bacteroides vulgatus, Bifidobacterium pseudocatenulatum, Bifidobacterium angulatum,
  • the plurality of bacteria low comprise R. albus and L. acidophilus.
  • Table 1 Bacteria Relevant to Cancer Lactobacillus acidophilus Bifidobacterium angulatum Ruminococcus albus Bifidobacterium bifidum Ruminococcus gnavus Campylobacter hominis Dorea formicigenerans Clostridium leptum Eubacterium siraeum Clostridium methylpentosum Bacteroides stercoris Coprococcus catus Parabacteroides distasonis Eubacterium ventriosum Bifidobacterium breve Ruminococcus bromii Lactobacillus delbrueckii Victivallis vadensis Odoribacter splanchnicus Bacteroides eggerthii Akkermansia mu
  • the method further comprises obtaining such a blood sample from the subject, and identifying bacterial-tumor antigen (BAC-TA) cross-reactive memory T-cells detected in the sample.
  • the composition is administered weekly for six weeks. In some embodiments, the composition is administered twice a week. In some embodiments, the composition is administered twice a month. In some embodiments, the composition is administered monthly. In some embodiments, the administration continues for at least two months. In some embodiments, the administration continues for at least 6, 8, 12, or 24 months.
  • the effective amount is sufficient to effect a two-fold increase in gut microbiome bacteria low , and/or a significant decrease in bacterial-tumor antigen (BAC- TA) cross-reactive memory T-cells, in a biological sample obtained from the subject.
  • the method enhances efficacy of a cancer treatment.
  • the cancer treatment comprises immunotherapy and/or chemotherapy.
  • the immunotherapy comprises administration of immune checkpoint inhibitor therapy, vaccine therapy, cytokine therapy, and/or oncolytic virus therapy.
  • the chemotherapy comprises administration of paclitaxel.
  • Other chemotherapeutic agents, as well as other immunotherapeutic agents, known to those skilled in the art are likewise contemplated.
  • chemotherapeutic agents include, but are not limited to, cisplatin, carboplatin, pemtrexed, Adriamycin, Cytoxan.
  • immunotherapeutic agents include, but are not limited to, monoclonal antibodies, such as, for example, anti-HER2, anti-EGFR, and anti-VEGF.
  • the composition is administered prior to and/or concomitantly with the immunotherapy or chemotherapy.
  • the method enhances a type 1 immune response.
  • the method increases the relative amount of bacteria low that can be detected in a biological sample obtained from the subject. Examples of biological samples include blood and stool samples.
  • the composition comprises 1-100 x10 9 CFU of bacteria low . In some embodiments, the composition comprises 1-50 x10 9 CFU of bacteria low . In some embodiments, the composition comprises 1-10 x10 9 CFU of bacteria low . In some embodiments, the composition comprises 1-5 x10 9 CFU of bacteria low .
  • the administration of the composition is oral, rectal, or intragastric. In some embodiments, the composition is delivered in the form of a suppository. [0096] Also described is a composition comprising a plurality of bacteria that exhibit less than 50% homology to non-mutated tumor antigens (bacteria low ).
  • the plurality of bacteria low are selected from: Lactobacillus acidophilus, Ruminococcus albus, Ruminococcus gnavus, Dorea formicigenerans, Eubacterium siraeum, Bacteroides stercoris, Parabacteroides distasonis, Bifidobacterium breve, Lactobacillus delbrueckii, Odoribacter splanchnicus, Akkermansia muciniphila, Desulfovibrio piger, Mitsuokella multacida, Bifidobacterium catenulatum, Staphylococcus saprophyticus, Bacteroides vulgatus, Bifidobacterium pseudocatenulatum, Bifidobacterium angulatum, Bifidobacterium bifidum, Campylobacter hominis, Clostridium leptum, Clostridium methylpentosum, Coprococcus catus, Eubacterium ventrios
  • the plurality of bacteria low comprise R. albus and L. acidophilus.
  • the composition is substantially free of bacteria that exhibit greater than 50% homology to tumor antigens.
  • the plurality of bacteria high are selected from: Escherichia coli, Enterococcus faecalis, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas aeruginosa, Shigella dysenteriae, Enterobacter cloacae, Lactococcus lactis, Clostridium perfringens, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecium, Rhodopseudomonas palustris, Shigella boydii, Shigella sonnei, Finegoldia magna, Clostridium asparagiforme, Collinsella aerofaciens, Fusobacterium ulcerans, Aerococcus viridans, Bacteroides ovatus, Clostridium botulinum, Enterococcus gallinarum, Eubacterium limosum, Faecalibacter
  • the plurality of bacteria high exhibit at least 50% homology to self-proteins identified in a blood and/or stool sample obtained from the subject.
  • Table 2 Bacteria Relevant to Autoimmunity / Inflammatory Conditions Escherichia coli Collinsella aerofaciens Enterococcus faecalis Fusobacterium ulcerans Pseudomonas fluorescens Aerococcus viridans Pseudomonas putida Bacteroides ovatus Pseudomonas aeruginosa Clostridium botulinum Shigella dysenteriae Enterococcus gallinarum Enterobacter cloacae Eubacterium limosum Lactococcus lactis Faecalibacterium prausnitzii Clostridium perfringens Holdemania filiformis Klebsiella pneumoniae Lactobacillus plantarum Staphylococcus aureus Lactobacillus reuteri Entero
  • the composition is administered weekly for at least one year. In some embodiments, the composition is administered twice a week. In some embodiments, the composition is administered twice a month. In some embodiments, the composition is administered monthly. In some embodiments, the administration continues for at least two months. In some embodiments, the administration continues for at least 6, 8, 12, or 24 months. [0101] In some embodiments, the effective amount is sufficient to effect a two-fold increase in gut microbiome bacteria high in a biological sample obtained from the subject. Examples of biological samples include blood and stool samples. In some embodiments, the method further comprises obtaining a biological sample from the subject, and identifying self-protein reactive memory T-cells. [0102] In some embodiments, the method elicits a type 2 immune response.
  • the method elicits an increase in the amount of IL-10 and/or IL-6 that can be detected in a biological sample obtained from the subject.
  • the composition 1-100 x10 9 CFU of bacteria high .
  • the composition comprises 1-50 x10 9 CFU of bacteria high .
  • the composition comprises 1-10 x10 9 CFU of bacteria high .
  • the composition comprises 1-5 x10 9 CFU of bacteria high .
  • the administration of the composition is oral, rectal, or intragastric.
  • the composition is delivered in the form of a suppository.
  • composition comprising a plurality of bacteria that exhibit at least 50% amino acid sequence homology to self-proteins (bacteria high ).
  • the plurality of bacteria high are selected from: Escherichia coli, Enterococcus faecalis, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas aeruginosa, Shigella dysenteriae, Enterobacter cloacae, Lactococcus lactis, Clostridium perfringens, Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecium, Rhodopseudomonas palustris, Shigella boydii, Shigella sonnei, Finegoldia magna, Clostridium asparagiforme, Collinsella aerofaciens, Fusobacterium ulcerans, Aerococcus viridans
  • the plurality comprises at least four species of bacteria high . In some embodiments, the plurality comprises 6-10 species of bacteria high .
  • Pharmaceutical compositions, Administration, and Dosage [0108] In some embodiments, the subject to be treated in the above methods is human. In some embodiments, the subject is suspected of having, or has been diagnosed with cancer, such as breast cancer, or an autoimmune disease. Determination of amounts and appropriate means of administration are determined under the guidance of a treating physician, taking into account an individual patient’s condition.
  • Pharmaceutical compositions disclosed herein include one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; peptides; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, phosphate buffered saline and the like
  • carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins e.g., aluminum hydroxide
  • preservatives e.g., aluminum hydroxide
  • compositions and/or bacteria disclosed herein are administered in any suitable manner, often with pharmaceutically acceptable carriers. Suitable methods of administering compositions in the context of the present invention to a subject are available, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • Compositions of the disclosure may be formulated
  • the dose administered to a patient should be sufficient to result in a beneficial therapeutic response in the patient over time, or to inhibit disease progression.
  • the composition is administered to a subject in an amount sufficient to elicit an effective response and/or to alleviate, reduce, cure or at least partially arrest symptoms and/or complications from the disease.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.”
  • Routes, order and/or frequency of administration of the therapeutic compositions disclosed herein, as well as dosage will vary from individual to individual, and may be readily established using standard techniques. In general, an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit.
  • Example 1 BAC-TA cross-reactive memory T-cells are present in the peripheral blood of volunteer donors and breast cancer patients.
  • CD4 T-cell epitope identification includes identifying putative Class II interacting sequences via a multi-algorithm approach to ensure responsiveness across diverse HLA DR alleles followed by functional phenotyping of the Th response (3).
  • T-cell responses were class II restricted for the peptide, bacterial, and recombinant oncogenic protein antigens.
  • BAC-TA T-cells can traffic to tumor and accelerate mammary cancer growth.
  • BAC-TA cross-reactive T-cells can also be identified in the spleens of mice (FVB/n strain). The median level of circulating YB1-p82 specific IL-10 secreting T-cells was 1:10,000 (range 1:10,000-1:71,000) of splenocytes from 30% of murine donors.
  • mice Sixty percent of the mice responded to BIRC5-p13 by secreting IL-10 at a median precursor frequency of 1:7000 (range 1:4000-1:76,000).
  • the median precursor frequency of Six1-p88 specific IL-10 secreting T-cells was 1:17,000 (range 1:13,000-1:20,000) splenocytes from 30% of donors (Fig.2A).
  • SPF specific-pathogen free
  • M6 syngeneic tumor cell line
  • C3T TgC3(1)-Tag
  • TIL tumor infiltrating lymphocytes
  • BAC-TA cross-reactive T-cells are found in spontaneous mammary tumors developing in the C3T mouse. We allowed mice to develop spontaneous tumors to a size of ⁇ 500 ⁇ 75 mm3. The tumors were then excised and enzymatically dissociated using the mouse TDK kit in combination with gentleMACs (Miltenyi Biotec). TIL were isolated via gradient centrifugation.
  • TIL were enriched for IL-10-secreting T- cells specific for bacteria that share >50% homology with Th2-selective epitopes from tumor associated antigens expressed in the C3T spontaneous tumors (P. aeruginosa/70% homology with YB1 epitope, E. coli/70% homology with YB1 epitope, and M. aurum/80% homology with PRL3 epitope) as compared to the bacterial specific T-cell levels in spleen (p ⁇ 0.01 for all).
  • P. aeruginosa/70% homology with YB1 epitope E. coli/70% homology with YB1 epitope
  • M. aurum/80% homology with PRL3 epitope M. aurum/80% homology with PRL3 epitope
  • mice received weekly (for 6 weeks) oral treatments of 1x10 9 CFU of a combination of L. acidophilus and R. Albus, both bacteria low species or a combination of E.coli and P. aeruginosa, both bacteria high species.
  • Fig.4C the control
  • Th phenotype for the IGFBP-2 protein (3) The majority of the IFN-g inducing Th1 selective epitopes are found in the N-terminus of the protein (aa 1-163). A vaccine consisting of the N-terminus could inhibit tumor growth. The C-terminus of IGFBP-2, in contrast, was enriched with Th2 selective epitopes secreting high levels of IL-10. A C-terminus vaccine (aa 164-328), when used for immunization, had no effect on tumor growth.
  • IFN-g ELISPOT Two weeks after the last vaccine, antigen specific immunity was measured by IFN-g ELISPOT (Fig.5).
  • Example 4 Precision modulation of the gut microbiome with bacteria low can limit tumor growth in a transgenic mammary tumor model of triple negative breast cancer and improve response to immunotherapy.
  • the C3T reflects a basal triple negative breast cancer phenotype, tumors express PD-L1, and the model is in an FVB/n background.
  • assays and microbiome data are relevant to this model.
  • C3T has an aggressive tumor that is resistant to both immune and chemotherapy (Fig.6).
  • C3T mice were implanted with the syngeneic basal breast cancer cell line (M6) then treated i.p. with anti-PD-L1 antibody (200ug) three days after implantation then twice weekly until the termination of the study (Fig.6A).
  • Paclitaxel therapy was given i.v. at 10mg/kg one day after implantation and 3 times per week until the termination of the study (Fig.6B).
  • treatment with bacteria low can limit tumor growth in C3T spontaneous mammary tumor model.
  • C3T develop tumors at a mean age of 18.5 ⁇ 2.83 weeks and the TIL express predominantly Gata 3, a marker of Th2 (12).
  • Mice were treated with bacteria starting at 6 weeks of age or sham gavage at which time the animals were observed for tumor development two to three times per week until sacrifice.
  • a fine needle aspiration (FNA) was taken every two weeks once tumors arose to monitor changes in the immune infiltrate and TCRVb consistent with BAC-TA T-cells over time.
  • FNA fine needle aspiration
  • Tumor growth kinetics were calculated by determining the change in volume between subsequent measurements and dividing by the number of days between the measurements, resulting in a rate value of mm 3 /day.
  • An initial assessment of the T-cell clonality of the tumor includes the evaluation of the Shannon index, which is a quantitative measure that reflects diversity. Lower values indicate more diversity while higher values indicate less diversity, which would suggest the development of specific T-cell clones in the tumor.
  • Shannon index is a quantitative measure that reflects diversity. Lower values indicate more diversity while higher values indicate less diversity, which would suggest the development of specific T-cell clones in the tumor.
  • mice will be implanted with the syngeneic tumor cell line (M6) in a manner similar to that described in Figure 6 and 24 hours later, animals will be treated with an anti-PD-L1 antibody (200ug/mouse, injected i.p. every other day for a total of 5 treatments). Appropriate IgG controls will also be employed. Tumor growth will be evaluated every 2-3 days and volumes calculated. Cohorts will be sacrificed and analyzed when the control tumor reach a volume close to 1000 ⁇ 200 mm 3 . We anticipate we will observe a 25% reduction in tumor growth in bacteria low treated mice as compared to controls evaluated in 15 mice/group. In addition, at sacrifice, the levels of BAC- TA cross-reactive T-cells can be assessed via IL-10 ELISPOT.
  • Example 5 Bacteria specific IL-10 secreting T-cells derived from the gut are cross-reactive with tumor antigens and accelerate tumor growth in mouse models [0129] This Example demonstrates that tumor antigen epitopes with elevated IL-10- secreting T-cell precursor frequencies share high sequence homology with epitopes from bacteria in the gut microbiome. Moreover, human T-cell lines generated from IL-10-inducing tumor antigen epitopes were cross-reactive to bacteria with high sequence homology, expressed cytokines and genes associated with intestinal intraepithelial lymphocytes and were of a memory phenotype.
  • Human PBMC or mouse splenocytes were evaluated by ELISPOT for antigen-specific IFN-gamma ( ⁇ ) using 10ug/ml peptides or IL-10 using 10 ⁇ g/ml peptides, 1 ⁇ g/ml human recombinant protein (Abnova), low endotoxin bovine serum albumin (BSA) (Gemini Bio-products), and 0.1 mg/ml low endotoxin bacterial lysate production as previously described [3]. All bacterial isolates were purchased from ATCC and cultured using methods established at ATCC. Cultures in log phase were lysed in sterile PBS with five rounds of freezing and thawing.
  • the antigen-specific T-cells were a mean of 75% (range 66-90%) CD3+ T- cells, which consisted predominantly of CD4+ T-cells (mean 65%, range 58-78% of CD3+), with few CD8+ T-cells (mean 10%; range, 8-12% of CD3+).
  • Splenocytes were cultured with a previously optimized dose of P. aeruginosa lysate (0.1 ⁇ g/ml) that had been treated to reduce endotoxin. On day 5 of culture, 20 ng/ml recombinant murine IL-2 was added. On day 8 of culture, autologous, irradiated splenocytes loaded with P.
  • aeruginosa lysate was added 1:1 to the original T-cell culture.
  • the P. aeruginosa specific T-cells were a mean of 75% (range 66-90%) CD3 + T-cells, which consisted predominantly of CD4 + T-cells (mean 65%, range 58-78% of CD3 + ), with few CD8 + T-cells (mean 10%; range, 8-12% of CD3 + ).
  • Mutation location data was obtained from the Exome Variant Server, NHLBI GO Exome Sequencing Project (ESP) Seattle, WA, data release (ESP6500SI-V2).
  • Homologous proteins for an epitope were obtained by querying the refseq protein database via Blast using the entire protein and filtering for bacterial, fungal and viral organisms in the human microbiome as defined in the Human Microbiome Project [5].
  • the Blast search settings were set with a maximum E-value of 200000 and a maximum of 10000 hits; all other search settings were default. Epitopes were not evaluated further if there was ⁇ 50% identity with any microorgnisms [6].
  • Murine Study Design [0137] Animal care and use were in accordance with University of Washington IACUC guidelines.
  • mice were randomized into treatment groups by age and assigned sequentially until the study was fully enrolled. The investigators and animal caretakers were not blinded to the treatment groups, but the treatments were administered in random cage order. In vivo studies were terminated when the volume of the treated group was statistically significantly more than the control for at least two measurements. All mice in in vivo experiments were included in the data analysis. All methods are reported in accordance with ARRIVE guidelines for animal studies. [0138] Animal model, adoptive transfer and assessment of tumor growth.
  • FVB mice Female, FVB mice (8 weeks old, median weight: 18 g, range: 16.5-18.5, Jackson Labs) FVB-Tg(C3-1- TAg)cJeg/Jeg (C3(1)-Tag) mice (6 weeks old; median weight: 18.5 g, range: 16.5-18.5 g; provided by Dr. Jeff Green, NCI) were used in this study. Tumors derived from the C3(1)Tag mouse are consistent with a basal phenotype [7].
  • the mouse mammary tumor cell line, M6 is derived from a spontaneous tumor from C3(1)-Tag mice (provided by Dr. Jeff Green, NCI [8]).
  • the M6 cell line was authenticated before use and verified to express the SV40 antigen by Western blot and negligible levels of estrogen receptor by RT-PCR.
  • 0.5 x 10 6 M6 cells were implanted into the flank [3]. Some mice were allowed to develop spontaneous tumors to a volume of 500 ⁇ 50 mm 3 . Tumors were measured as previously described [2]. All tumor growth is presented as mean tumor volume (mm 3 ⁇ SEM). [0139] For adoptive transfer, on day 14 of T-cell culture, 1X10 7 P.
  • aeruginosa-specific T- cells or na ⁇ ve splenocytes were labeled with 320 ⁇ g/ml XenoLight DiR (Perkin Elmer) in 5ml PBS then washed twice with PBS. Uptake of the dye was confirmed via flow cytometry. The cells were injected into the tail vein of each of 4-5 mice bearing 25-75mm 3 tumors. After 72h, mice were fluorescently imaged on a Perkin Elmer IVIS imager and fluorescent output (Total flux, photons/second) was quantified on the Living Image software. [0140] Cell phenotyping. RNA was isolated from human and murine T cells using the RNAqueous-4PCR (Life Technologies) kit according to manufacturer’s instructions.
  • RNA quantity was determined with a NanoDrop Spectrophotometer.
  • cDNA was synthesized from 100 pg of RNA using the SuperScript III RT (Life Technologies) kit according to the manufacturer’s instructions.
  • Gene expression was assessed via TaqMan (ABI 7900HT) Real time PCR using 50 ng of cDNA and 1pg of the specific TaqMan Gene Expression Array (Life Technologies).
  • Cytokine secretion was measured with the Milliplex Multiplex Assays using Luminex (Millipore Sigma) according to the manufacturer’s instructions in triplicates from 72h cell culture supernatants.
  • Intestines were then cut into 0.5-cm pieces, transferred into 50 ml conical tubes, and shaken at 250 rpm for 30 min at 37°C in Hanks’-5 (Ca/Mg-free HBSS plus 5% FBS) with 1 mM EDTA and 1 mM DTT (Sigma-Aldrich). The cell suspensions were passed through a strainer, then over sterile gauze.
  • T-cell receptor beta chain CDR3 regions were sequenced from DNA isolated from murine intestinal intraepithelial lymphocytes, tumor infiltrating lymphocytes and spleen or human T-cell lines, PBMC or tumor biopsies by ImmunoSeq (Adaptive Biotechnologies), with primers annealing to V and J segments, resulting in amplification of rearranged VDJ segments from each cell and differential expression analyzed with the ImmunoSeq Analyzer software. [0145] 16s Amplicon Sequencing and Analysis. Frozen tumor and spleen were provided to Microbiome Insights, Inc.
  • the library was preparation using the high-fidelity Phusion polymerase for amplification of 16s marker genes.
  • the PCR reactions were normalized using the high-throughput SequalPrep 96-well Plate Kit. Samples are then pooled to make one library that was quantified accurately with the KAPA qPCR Library Quant kit. Library pools were sequenced on the Illumina MiSeq using the corresponding primer sets for prokaryotic regions. [0146] Statistical analysis. R version 3.3.0 was used for all statistical analyses. ELISPOT count data was pre-processed according to published methods [9].
  • Tumor antigen epitopes with elevated IL-10-secreting T-cell precursor frequencies share high sequence homology with epitopes from bacteria in the gut microbiome.
  • 11 bacterial species with homologies to more than ten tumor antigen epitopes with at least 86% of the donors evaluated responding to those epitopes by secreting IL-10. Table 4). [0150] Table 4.
  • BAC-TA IL-10 stimulating CD4 T-cell epitopes that are commonly detected in the majority of individuals tested.
  • Human T-cell lines generated from IL-10-inducing tumor antigen epitopes were cross-reactive to bacteria with high sequence homology, expressed cytokines and genes associated with intestinal intraepithelial lymphocytes and were of a memory phenotype.
  • Three different IL-10-selective epitopes with a high IL-10-secreting precursor frequency of antigen-specific T-cells in PBMC were used for evaluation.
  • the median level of circulating Yb1-p82 specific IL-10 secreting T-cells was 1:56,000 (range 1:600-1:80,000) of PBMC from 40% of donors.
  • Antigen-specific T-cell lines were generated with each of these epitopes.
  • Yb1-p82 is 58% homologous to Pseudomonas (P.) aeruginosa and shares no homology with Ruminococcus (R.) albus.
  • PRL3-p104- and BIRC5-p13- specific T-cells demonstrated significant IL-10 secretion when incubated with M. aurum lysate (63% homologous; mean, 68.3 ⁇ 19 CSPW) as compared to the R.
  • the bacterial-tumor antigen cross reactive epitopes generated IL-10 secretion from the T-cells in all donors (mean, 1,954 pg/ml; range 1,000- 2,705 pg/ml), but very low levels of IL-4 (mean 10 pg/ml) and IL-5 (95 pg/ml).
  • the IL-10 inducing epitopes also secreted high levels of IL-6 (mean, 41,510 pg/ml; range 38,812- 45,272 pg/ml) and higher levels of TNF- ⁇ (mean, 28,442 pg/ml; range, 26,062-29,797 pg/ml), IL1- ⁇ (mean, 12,791 pg/ml, range 12, 505-12,965 pg/ml) as compared to autologous CD3+ T-cells (Fig.9D).
  • the genes c-Maf, PD1, ICOS, FOXP3, CCR9, ROR-g, LAG3 and CTLA4 were expressed greater than a mean of 4-fold in the bacterial-tumor antigen cross reactive T-cells as compared to autologous unstimulated CD3 + selected T-cells.
  • AHR, ThPOK and GATA3 were expressed at lower levels in the T-cell lines (Fig.9E), suggesting an intestinal intraepithelial-like lymphocyte phenotype.
  • the median level of circulating Yb1-p82 specific IL-10 secreting T-cells was 1:10,000 (range 1:10,000-1:71,000) of splenocytes from 30% of donors. No PRL3-p104-specific IL-10- secreting cells were observed in any donor. Sixty percent of the mice responded to BIRC5- p13 by secreting IL-10 at a median precursor frequency of 1:7000 (range 1:4000-1:76,000). [0155] Yb1-p82-specific T-cells confirmed to be epitope and protein specific exhibited significant IL-10 secretion when incubated with P. aeruginosa lysate (mean, 113.5 ⁇ 18 CSPW) as compared to the negative lysate control R.
  • the IL-10 inducing epitopes also secreted high levels of IL-6 (mean 3022 pg/ml) and higher levels of IL-10 (mean, 175 pg/ml), IL-5 (mean 2248 pg/ml) and IL-13 (mean 785 pg/ml) as compared to autologous CD3+ T-cells (Fig.10C).
  • the genes c-Maf, PD1, FOXP3, ROR-g, and LAG3 were expressed greater than a mean of 20-fold in the bacterial-tumor antigen cross reactive T-cells as compared to unstimulated CD3 + selected T-cells.
  • AHR, ThPOK, ICOS, GATA3 and CTLA4 were expressed at lower levels in the T-cell lines (Fig. 10D). Significantly more central and effector memory cells than na ⁇ ve cells in both T-cell compartments were observed in the bacterial-tumor antigen cross-reactive T-cells as compared to autologous unstimulated CD3 + selected T-cells (p ⁇ 0.05 for all; Fig.10E). [0157] Significantly more TCRb were common between intestinal intraepithelial lymphocytes and tumor infiltrating lymphocytes than splenocytes and tumor infiltrating lymphocytes.
  • TCRb sequences among corresponding intestinal intraepithelial lymphocytes, splenocytes and tumor infiltrating lymphocytes from mice who developed spontaneous breast cancer.
  • the median number of shared TCRb between intestinal intraepithelial lymphocytes and tumor infiltrating lymphocytes was 91 (range, 7- 1450) and between splenocytes and tumor infiltrating lymphocytes was 17 (range, 1-143; Fig 11A).
  • Pseudomonas aeruginosa-specific T-cells traffic to breast cancer and promote tumor growth. Since we had demonstrated that peptide-specific T-cells could cross-react with bacteria with high homology, we next questioned if bacterial-specific T-cells could cross- react with a high homology epitope. Using P. aeruginosa as a representative bacterium, we generated an antigen-specific T-cell line. P. aeruginosa-specific T-cells were cross-reactive with the high homology epitope Yb1-p82.
  • the P. aeruginosa-specific T-cells were specifically detected in the tumor and not observed in secondary lymphoid organs such as the spleen.
  • the TIL were specific for P. aeruginosa.
  • Antigen-specific T cells cross-reactive between IL-10-selective self-epitopes and bacteria were present in spontaneous breast cancer. Significantly more IL-10 secreting cells were detected in the TIL stimulated with lysate from bacteria with high homologies to tumor antigens, P. aeruginosa (380 ⁇ 47cSPW), E. coli (435 ⁇ 55 cSPW) or M.
  • TCRb sequences observed in the corresponding tumor biopsy two sequences were also found in the HIF1a-p312-specific T-cell line, CASSEEAGGYNEQFF (SEQ ID NO: 1; TCR096; representing 0.01% of the tumor TCRb repertoire) and CASSLEGSSNF (SEQ ID NO: 2; TCR123; representing 0.07% of the tumor TCRb repertoire; Fig.13D). None of the 11 TCRb sequences observed in the BIRC5-p13-specific T-cell line generated from Donor 1’s PBMC were found in the tumor biopsy of Donor 1. Nineteen distinct TCRb sequences were observed for the BIRC5-p13 T-cell line in Donor 2 (Fig 13E).

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  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des organismes spécifiques dérivés du microbiome intestinal, ne partageant que rarement ou jamais des homologues avec des antigènes tumoraux, qui sont utilisés pour moduler des lymphocytes T à réaction croisée d'antigènes tumoraux bactériens. Des bactéries présentant peu ou pas d'homologies peuvent être enrichies en toute sécurité dans l'intestin et peuvent empêcher ces lymphocytes T à réaction croisée de se multiplier. Cette modulation de précision du microbiome intestinal réduit la croissance tumorale et augmente l'efficacité d'une thérapie par inhibiteur de point de contrôle immunitaire dans un modèle de cancer du sein triple négatif. De plus, l'utilisation d'organismes spécifiques dérivés du microbiome intestinal qui partagent des homologies avec des auto-protéines peut être utilisée pour moduler la réponse inflammatoire auto-immune. Cette modulation de précision du microbiome intestinal peut être utilisée pour déclencher une réponse immunitaire de type 2.
PCT/US2023/078034 2022-10-31 2023-10-27 Peptides et probiotiques ciblés pour la réduction ou l'augmentation de lymphocytes t mémoires à réaction croisée d'auto-antigènes bactériens pour traiter le cancer ou une maladie auto-immune WO2024097608A2 (fr)

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MA41020A (fr) * 2014-11-25 2017-10-03 Evelo Biosciences Inc Compositions probiotiques et prébiotiques, et leurs procédés d'utilisation pour la modulation du microbiome

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